CN111394300B - Large-scale stem cell culture method - Google Patents
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Abstract
The invention provides a large-scale stem cell culture method, which comprises the following steps: swelling the 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter is 0.5-10mm, and the pore diameter of the microcapsule is 10nm-10000 nm; inoculating the single cell suspension into the swelled 3D microcapsule, wherein the inoculation density of the stem cells is 1-8 multiplied by 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: static culturing for 1-3 days, adjusting the stirring speed to 50-70RPM during static culturing, adjusting the pH to 7.0 and DO 50%, adjusting the temperature to 37 +/-1 ℃, supplementing the culture medium every 2 days, performing perfusion culturing for 3-5 days, increasing the perfusion flow rate from 0.8vvd to 2vvd during perfusion culturing, adjusting the stirring speed to 100RPM, and supplementing the culture medium every 2 days; 3D microcapsule dissolving agent is used for carrying out the capsulizing; harvesting the expanded cells; the large-scale stem cell culture method provided by the invention can solve the problem that cells are difficult to harvest in large-scale culture, and the stem cells cultured by the method have good proliferation effect, high survival rate and short harvesting time.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a large-scale stem cell culture method.
Background
Stem cells are primitive undifferentiated cells with multi-directional differentiation potential and self-replication ability, which are primitive cells forming various tissues and organs of mammals, and the use of stem cells is very wide, and relates to multiple fields of medicine.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a large-scale stem cell culture method.
The specific technical scheme of the invention is as follows:
the invention provides a large-scale stem cell culture method, which comprises the following steps:
swelling the 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter is 0.5-10mm, and the pore diameter of the microcapsule is 10nm-10000 nm;
inoculating the single cell suspension into the swelled 3D microcapsule, wherein the inoculation density of the stem cells is 1-8 multiplied by 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: static culturing for 1-3 days at stirring speed of 50-70RPM, pH of 7.0 and DO of 50% and temperature of 37 + -1 deg.C, and supplementing every 2 daysCulturing a culture medium, performing perfusion culture for 3-5 days, increasing the perfusion flow rate from 0.8vvd to 2vvd during perfusion culture, adjusting the rising speed of the perfusion flow rate to 1-3vvd/h, adjusting the stirring speed to 100rpm, and supplementing the culture medium every 2 days;
3D microcapsule dissolving agent is used for carrying out the capsulizing;
and harvesting the expanded cells.
The 3D microcapsule provided by the invention supports the efficient production and harvesting of most cell types such as multiple attachment-dependent cells (adherent cells) and facultative adherent cells, and comprises the following steps: the invention relates to a method for harvesting fibroblast cells (endothelial cells, mesenchymal cells, osteoblasts, myocardial cells, chondrocytes and the like), epithelial cells (skin cells, epidermal derivatives, digestive tract epithelial cells and the like), migratory cells (macrophages, tumor cells and the like), polymorphic cells (neuronal cells, glial cells and the like) and the like, wherein the harvesting method of the amplified cells is determined according to specific conditions, and if ATF2 cell interception equipment is adopted for harvesting, the method can improve the cell yield during large-scale cell culture.
Further, the 3D microcapsule after swelling is inoculated with the single cell suspension by adopting a rotary culture mode, wherein the stirring speed of the rotary culture is 30-50 r/min.
Further, the specific operation of swelling the 3D microcapsule in step (2) is as follows: stirring the 3D microcapsules and a DMEM/F12 culture medium uniformly, keeping the concentration of the 3D microcapsules in the DMEM/F12 culture medium at 100-1000g/L, standing for 3-5h, and transferring the 3D microcapsules into a DMEM/F12 culture medium containing 10% FBS, wherein the volume ratio of the DMEM/F12 culture medium containing 10% FBS to the DMEM/F12 culture medium is 1-2: 1-2.
Further, the preparation method of the 3D microcapsule comprises the following steps:
s1: uniformly mixing 1-6 wt% of sodium alginate solution and 0.1-5 wt% of gelatin solution in a ratio of 0.1-2:0.1-2 to prepare a first mixed solution;
s2: adding a foaming agent into the first mixed solution, and uniformly mixing, wherein the concentration of the foaming agent in the first mixed solution is 5-100g/L, so as to prepare a second mixed solution;
s3: dripping CaCl into the second mixed solution2Solution of CaCl2The volume ratio of the solution to the first mixed solution is 1-2:1-2, and a 3D microcapsule solution is prepared;
s4: washing the 3D microcapsule solution with normal saline for 3-6 times, and then carrying out vacuum drying to obtain a dry 3D microcapsule;
s5: and flattening and sterilizing the dried 3D microcapsule to obtain the 3D microcapsule.
Further, the 3D microcapsule dissolving agent comprises amino acid + citric acid + sodium citrate + EDTA sodium salt in a mass ratio of 0.1-1.0:0.1-2.0:0.1-1.0:0.1-2.0, wherein the amino acid comprises any one of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine and histidine.
Further, the volume ratio of the using amount of the dissolving agent to the swelled 3D microcapsule is 1-2: 1-4.
The invention also provides a microcapsule for large-scale stem cell culture, and the preparation method of the 3D microcapsule comprises the following steps:
s1: uniformly mixing 1-6 wt% of sodium alginate solution and 0.1-5 wt% of gelatin solution in a ratio of 0.1-2:0.1-2 to prepare a first mixed solution;
s2: adding a foaming agent into the first mixed solution, and uniformly mixing, wherein the concentration of the foaming agent in the first mixed solution is 5-100g/L, so as to prepare a second mixed solution;
s3: dripping CaCl into the second mixed solution2Solution of said CaCl2The volume ratio of the solution to the first mixed solution is 1-2:1-2, and a 3D microcapsule solution is prepared;
s4: washing the 3D microcapsule solution with normal saline for 3-6 times, and then carrying out vacuum drying to obtain a dried 3D microcapsule;
s5: flattening and sterilizing the dried 3D microcapsule to obtain the 3D microcapsule.
The invention also provides large-scale stem cell culture equipment, which comprises a bioreactor, a hollow fiber filter, a control device and a waste liquid bottle, wherein the control device comprises a control host, a magnetic suspension centrifugal sample injection pump and a filtrate/liquid supplement auxiliary pump which are integrated into a whole, the hollow fiber filter is used for filtering waste liquid and intercepting cells in the bioreactor, the bioreactor is respectively connected with a cell containing bottle, a culture medium containing bottle, a 3D microcapsule containing bottle and a lytic agent containing bottle through a first connecting pipe and is connected with the central control fiber filter through a sample injection pipeline and a return pipeline, the hollow fiber filter is connected with the magnetic suspension centrifugal sample injection pump and the filtrate/liquid supplement auxiliary pump through a second connecting pipe, and the magnetic suspension centrifugal sample injection pump is connected with the waste liquid bottle through a waste liquid pipe.
Furthermore, the hollow fiber filter comprises a filter body and an ultrasonic reflux flowmeter arranged on the filter body, the bottom end of the filter body is connected with a sample injection pipeline of a sample injection pressure sensor through a magnetic suspension pump head respectively, a reflux pressure sensor and an aseptic sampling fracture are sequentially arranged on a reflux pipeline close to the filter body, and the sample injection pressure sensor and the reflux pressure sensor are connected with a control host.
And furthermore, a filtrate pressure sensor is arranged at one end, close to the filter body, of a second connecting pipe connected with the magnetic suspension centrifugal sample injection pump, and the filtrate pressure sensor is connected with the control host.
Further, the bioreactor is also connected with an automatic filling machine.
The method for culturing the stem cells on a large scale can solve the problem that the stem cells are difficult to harvest when cultured on a large scale in the prior art, and the stem cells cultured by the method provided by the invention have the advantages of good proliferation effect, high survival rate and short harvesting time.
Drawings
FIG. 1 is a schematic view showing the construction of a large-scale stem cell culturing apparatus according to examples 1 to 3;
the device comprises a bioreactor 1, a control device 2, a waste liquid bottle 3, a sample injection pipeline 4, a return pipeline 5, a second connecting pipe 6, a waste liquid pipe 7, a filter body 8, an ultrasonic return flow meter 9, a magnetic suspension pump head 10, a sample injection pressure sensor 11, a sample injection pressure sensor 12, a return pressure sensor 13 and a filtrate pressure sensor 14.
Detailed Description
Example 1
This example provides a large scale stem cell culture process, which is carried out by the apparatus shown in FIG. 1; the culture method comprises the following steps: swelling the 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter is 0.5, and the pore diameter of the microcapsule is 10 nm;
inoculating mesenchymal stem cells into the swelled 3D microcapsules, wherein the inoculation density of the stem cells is 1 multiplied by 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: performing static culture for 1 day, adjusting the stirring speed to 50RPM, adjusting the pH to 7.0 and DO 50% and the temperature to 37 ℃ during the static culture, supplementing the culture medium every 2 days, performing perfusion culture for 3 days, increasing the perfusion flow rate from 0.8vvd to 2vvd during the perfusion culture, adjusting the rising speed of the perfusion flow rate to 1vvd/h, adjusting the stirring speed to 100RPM, and supplementing the culture medium every 2 days;
3D microcapsule dissolving agent is used for carrying out the capsulizing;
harvesting the expanded cells;
inoculating the single cell suspension to the swelled 3D microcapsule by adopting a rotary culture mode, wherein the stirring speed of the rotary culture is 30 r/min;
the specific operation of swelling the 3D microcapsules is as follows: uniformly stirring the 3D microcapsules and a DMEM/F12 culture medium, keeping the concentration of the 3D microcapsules in the DMEM/F12 culture medium at 100g/L, standing for 3h, transferring the 3D microcapsules into a DMEM/F12 culture medium containing 10% FBS, wherein the volume ratio of the DMEM/F12 culture medium containing 10% FBS to the DMEM/F12 culture medium is 1: 2;
the preparation method of the 3D microcapsule comprises the following steps:
s1: uniformly mixing a sodium alginate solution with the weight percentage of 1 wt% and a gelatin solution with the weight percentage of 0.1 wt% in a ratio of 0.1:2 to prepare a first mixed solution;
s2: adding a foaming agent into the first mixed solution, and uniformly mixing, wherein the concentration of the foaming agent in the first mixed solution is 5g/L, so as to prepare a second mixed solution;
s3: dripping CaCl into the second mixed solution2Solution of said CaCl2The volume ratio of the solution to the first mixed solution is 2:1, and a 3D microcapsule solution is prepared;
s4: washing the 3D microcapsule solution with normal saline for 3 times, and then carrying out vacuum drying to obtain a dried 3D microcapsule;
s5: flattening and sterilizing the dried 3D microcapsule to obtain a 3D microcapsule;
the 3D microcapsule dissolving agent comprises serine, citric acid, sodium citrate and EDTA sodium salt in a mass ratio of 1:2:1: 2;
the volume ratio of the using amount of the dissolving agent to the swelled 3D microcapsule is 2: 1;
wherein, the device shown in figure 1 comprises a bioreactor 1, a hollow fiber filter, a control device 2 and a waste liquid bottle 3, the control device comprises a control host, a magnetic suspension centrifugal sample injection pump and a filtrate/liquid supplement auxiliary pump which are integrated into a whole, the hollow fiber filter is used for filtering waste liquid and intercepting cells in the bioreactor, the bioreactor is respectively connected with a cell containing bottle, a culture medium containing bottle, a 3D microcapsule containing bottle and a dissolving agent containing bottle through a first connecting pipe, not shown in the figure and is connected with a central control fiber filter through a sample introduction pipeline 4 and a return pipeline 5, the hollow fiber filter is connected with the magnetic suspension centrifugal sample injection pump and the filtrate/liquid supplement auxiliary pump through a second connecting pipe 6, the magnetic suspension centrifugal sample injection pump is connected with the waste liquid bottle 3 through a waste liquid pipe 7, the hollow fiber filter comprises a filter body 8 and an ultrasonic return flow meter 9 arranged on the filter body, the bottom of filter body is continuous with appearance pressure sensor 11 appearance pipeline 4 through magnetic suspension pump head 10 respectively, be close to being equipped with backward flow pressure sensor 12 and aseptic sample fracture 13 on the return line 5 of filter body 8 in proper order, appearance pressure sensor 11 and backward flow pressure sensor 12 are connected with the control host computer, the one end that is close to filter body 8 of second connecting pipe 6 that is connected with magnetic suspension centrifugal sample pump is equipped with filtrating pressure sensor 14, filtrating pressure sensor 14 is connected with the control host computer, bioreactor 1 still is connected with the automatic filling machine, not shown in the figure.
When the device and the method are used for cell culture, the microcarrier is firstly swelled, and the specific operation of swelling the 3D microcapsule is as follows: stirring 500g of 3D microcapsule and 1L of DMEM/F12 culture medium uniformly, keeping the concentration of the 3D microcapsule in the DMEM/F12 culture medium at 100g/L, standing for 3h, transferring the 3D microcapsule to 2L of DMEM/F12 culture medium containing 10% FBS, placing the swelled microcapsule into a 3D microcapsule containing bottle, and adding the solution containing 20 multiplied by 108Placing single cell suspension of each cell into a cell accommodating bottle, placing 2L DMEM/F12 culture medium into a culture medium accommodating bottle, placing 3D microcapsule dissolving agent into the dissolving agent accommodating bottle, controlling a starting filtrate/fluid infusion auxiliary pump in the device, introducing the single cell suspension and the swollen 3D microcapsules in the cell accommodating bottle and the 3D microcapsule accommodating bottle into a 5L bioreactor through a first connecting pipe for inoculation, detecting whether inoculation is successful or not through aseptic sampling fracture sampling in the inoculation process, introducing DMEM/F12 culture medium in the culture medium accommodating bottle through the first connecting pipe for cell amplification culture after inoculation is successful, introducing the 3D microcapsule dissolving agent through the first connecting pipe for de-encapsulation after the cell amplification is completed, starting a magnetic suspension centrifugal sample introduction pump after de-encapsulation is successful, introducing the contents of the bioreactor into a filter body for circular filtration, and the waste liquid enters a waste liquid tank along the second connecting pipe and the waste liquid pipe through a filtering membrane in the filter body, the amplified cells are retained in the bioreactor, frozen stock solution is added into the amplified cells, then the cells can be guided into an automatic filling machine for filling, and the filled cells are stored in a liquid nitrogen environment.
Example 2
This example provides a large scale stem cell culture process, which is carried out by the apparatus shown in FIG. 1; the culture method comprises the following steps: swelling the 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter is 5mm, and the pore diameter of the microcapsule is 5000 nm;
inoculating the mesenchymal stem cells into the swelled 3D microcapsules, and dryingThe inoculation density of the cells was 4X 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: performing static culture for 2 days, adjusting the stirring speed to 60RPM, adjusting the pH to 7.0 and DO 50% and the temperature to 37 ℃ during the static culture, supplementing the culture medium every 2 days, performing perfusion culture for 4 days, increasing the perfusion flow rate from 0.8vvd to 2vvd during the perfusion culture, adjusting the rising speed of the perfusion flow rate to 2vvd/h, adjusting the stirring speed to 100RPM, and supplementing the culture medium every 2 days;
3D microcapsule dissolving agent is used for carrying out the capsulizing;
harvesting the expanded cells;
inoculating the single cell suspension to the swelled 3D microcapsule by adopting a rotary culture mode, wherein the stirring speed of the rotary culture is 40 r/min;
the specific operation of swelling the 3D microcapsules is as follows: uniformly stirring the 3D microcapsules and a DMEM/F12 culture medium, keeping the concentration of the 3D microcapsules in the DMEM/F12 culture medium at 500g/L, standing for 4h, transferring the 3D microcapsules into a DMEM/F12 culture medium containing 10% FBS, wherein the volume ratio of the DMEM/F12 culture medium containing 10% FBS to the DMEM/F12 culture medium is 1: 1;
the preparation method of the 3D microcapsule comprises the following steps:
s1: uniformly mixing a sodium alginate solution with the weight percentage of 3 wt% and a gelatin solution with the weight percentage of 3 wt% in a ratio of 1:1 to prepare a first mixed solution;
s2: adding a foaming agent into the first mixed solution, and uniformly mixing, wherein the concentration of the foaming agent in the first mixed solution is 50g/L, so as to prepare a second mixed solution;
s3: dripping CaCl into the second mixed solution2Solution of said CaCl2The volume ratio of the solution to the first mixed solution is 1:1, and a 3D microcapsule solution is prepared;
s4: washing the 3D microcapsule solution with normal saline for 5 times, and then carrying out vacuum drying to obtain a dry 3D microcapsule;
s5: flattening and sterilizing the dried 3D microcapsule to obtain a 3D microcapsule;
the 3D microcapsule dissolving agent comprises alanine, citric acid, sodium citrate and EDTA sodium salt in a mass ratio of 1.0:0.1:1.0: 0.1;
the volume ratio of the using amount of the dissolving agent to the swelled 3D microcapsule is 1: 1;
wherein, the device shown in figure 1 comprises a bioreactor 1, a hollow fiber filter, a control device 2 and a waste liquid bottle 3, the control device comprises a control host, a magnetic suspension centrifugal sample injection pump and a filtrate/liquid supplement auxiliary pump which are integrated into a whole, the hollow fiber filter is used for filtering waste liquid and intercepting cells in the bioreactor, the bioreactor is respectively connected with a cell containing bottle, a culture medium containing bottle, a 3D microcapsule containing bottle and a dissolving agent containing bottle through a first connecting pipe, not shown in the figure and is connected with a central control fiber filter through a sample introduction pipeline 4 and a return pipeline 5, the hollow fiber filter is connected with the magnetic suspension centrifugal sample injection pump and the filtrate/liquid supplement auxiliary pump through a second connecting pipe 6, the magnetic suspension centrifugal sample injection pump is connected with the waste liquid bottle 3 through a waste liquid pipe 7, the hollow fiber filter comprises a filter body 8 and an ultrasonic return flow meter 9 arranged on the filter body, the bottom of filter body is continuous with appearance pressure sensor 11 appearance pipeline 4 through magnetic suspension pump head 10 respectively, be close to being equipped with backward flow pressure sensor 12 and aseptic sample fracture 13 on the return line 5 of filter body 8 in proper order, appearance pressure sensor 11 and backward flow pressure sensor 12 are connected with the control host computer, the one end that is close to filter body 8 of second connecting pipe 6 that is connected with magnetic suspension centrifugal sample pump is equipped with filtrating pressure sensor 14, filtrating pressure sensor 14 is connected with the control host computer, bioreactor 1 still is connected with the automatic filling machine, not shown in the figure.
When the device and the method are used for cell culture, the microcarrier is firstly swelled, and the specific operation of swelling the 3D microcapsule is as follows: stirring 5000g of 3D microcapsule and 10L of DMEM/F12 culture medium uniformly, standing for 4h, transferring 3D microcapsule into 10L of 10% FBS-containing DMEM/F12 culture medium, placing the swelled microcapsule into a 3D microcapsule containing bottle, and placing the solution containing 20 × 109Placing single cell suspension of each cell into a cell accommodating bottle, placing 30L DMEM/F12 culture medium into the culture medium accommodating bottle, and placing 3D microcapsule dissolving agent into the dissolving bottleA lytic agent containing bottle, a filtrate/fluid infusion auxiliary pump in the control device is started, the single cell suspension and the swollen 3D microcapsules in the cell containing bottle and the 3D microcapsule containing bottle are led into a 50L bioreactor through a first connecting pipe for inoculation, whether the inoculation is successful or not can be detected by sampling through an aseptic sampling fracture in the inoculation process, after the inoculation is successful, DMEM/F12 culture medium led into the culture medium containing bottle through the first connecting pipe is used for carrying out amplification culture on the cells, after the amplification is finished, 3D microcapsule dissolving agent is led into the first connecting pipe for capsulizing, after the capsulizing is successful, a magnetic suspension centrifugal sample injection pump is started, the contents of the bioreactor are led into a filter body for circular filtration, waste liquid enters a waste liquid tank through a filtering membrane in the filter body along a second connecting pipe and a waste liquid pipe, the amplified cells are kept in the bioreactor, and adding the frozen stock solution into the amplified cells, introducing into an automatic filling machine for filling, and storing the filled cells in a liquid nitrogen environment.
Example 3
This example provides a large scale stem cell culture process, which is carried out by the apparatus shown in FIG. 1; the culture method comprises the following steps: swelling the 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter is 10mm, and the pore diameter of the microcapsule is 10000 nm;
inoculating the mesenchymal stem cells into the swelled 3D microcapsules, wherein the inoculation density of the stem cells is 8 multiplied by 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: performing static culture for 3 days, adjusting the stirring speed to 70RPM, adjusting the pH to 7.0 and DO 50% and the temperature to 37 ℃ during the static culture, supplementing the culture medium every 2 days, performing perfusion culture for 5 days, increasing the perfusion flow rate from 0.8vvd to 2vvd during the perfusion culture, adjusting the rising speed of the perfusion flow rate to 3vvd/h, adjusting the stirring speed to 100RPM, and supplementing the culture medium every 2 days;
3D microcapsule dissolving agent is used for carrying out the capsulizing;
harvesting the expanded cells;
inoculating the single cell suspension to the swelled 3D microcapsule by adopting a rotary culture mode, wherein the stirring speed of the rotary culture is 50 r/min;
the specific operation of swelling the 3D microcapsules is as follows: uniformly stirring the 3D microcapsules and a DMEM/F12 culture medium, keeping the concentration of the 3D microcapsules in the DMEM/F12 culture medium at 1000g/L, standing for 5h, transferring the 3D microcapsules into a DMEM/F12 culture medium containing 10% FBS, wherein the volume ratio of the DMEM/F12 culture medium containing 10% FBS to the DMEM/F12 culture medium is 1: 2;
the preparation method of the 3D microcapsule comprises the following steps:
s1: uniformly mixing a sodium alginate solution with the weight percentage of 6 wt% and a gelatin solution with the weight percentage of 5 wt% in a ratio of 0.1:2 to prepare a first mixed solution;
s2: adding a foaming agent into the first mixed solution, and uniformly mixing, wherein the concentration of the foaming agent in the first mixed solution is 100g/L, so as to prepare a second mixed solution;
s3: dripping CaCl into the second mixed solution2Solution of said CaCl2The volume ratio of the solution to the first mixed solution is 1:2, and a 3D microcapsule solution is prepared;
s4: washing the 3D microcapsule solution with normal saline for 6 times, and then carrying out vacuum drying to obtain a dry 3D microcapsule;
s5: flattening and sterilizing the dried 3D microcapsule to obtain a 3D microcapsule;
the 3D microcapsule dissolving agent comprises glycine, citric acid, sodium citrate and EDTA sodium salt in a mass ratio of 0.1:2.0:0.1: 2.0;
the volume ratio of the using amount of the dissolving agent to the swelled 3D microcapsule is 1: 4;
wherein, the device shown in figure 1 comprises a bioreactor 1, a hollow fiber filter, a control device 2 and a waste liquid bottle 3, the control device comprises a control host, a magnetic suspension centrifugal sample injection pump and a filtrate/liquid supplement auxiliary pump which are integrated into a whole, the hollow fiber filter is used for filtering waste liquid and intercepting cells in the bioreactor, the bioreactor is respectively connected with a cell containing bottle, a culture medium containing bottle, a 3D microcapsule containing bottle and a dissolving agent containing bottle through a first connecting pipe, not shown in the figure and is connected with a central control fiber filter through a sample introduction pipeline 4 and a return pipeline 5, the hollow fiber filter is connected with the magnetic suspension centrifugal sample injection pump and the filtrate/liquid supplement auxiliary pump through a second connecting pipe 6, the magnetic suspension centrifugal sample injection pump is connected with the waste liquid bottle 3 through a waste liquid pipe 7, the hollow fiber filter comprises a filter body 8 and an ultrasonic return flow meter 9 arranged on the filter body, the bottom of filter body is continuous with appearance pressure sensor 11 appearance pipeline 4 through magnetic suspension pump head 10 respectively, be close to being equipped with backward flow pressure sensor 12 and aseptic sample fracture 13 on the return line 5 of filter body 8 in proper order, appearance pressure sensor 11 and backward flow pressure sensor 12 are connected with the control host computer, the one end that is close to filter body 8 of second connecting pipe 6 that is connected with magnetic suspension centrifugal sample pump is equipped with filtrating pressure sensor 14, filtrating pressure sensor 14 is connected with the control host computer, bioreactor 1 still is connected with the automatic filling machine, not shown in the figure.
When the device and the method are used for cell culture, the microcarrier is firstly swelled, and the specific operation of swelling the 3D microcapsule is as follows: stirring 50000g of 3D microcapsule and 100L of DMEM/F12 culture medium uniformly, standing for 5h, transferring the 3D microcapsule into 50L of 10% FBS-containing DMEM/F12 culture medium, placing the swelled microcapsule into a 3D microcapsule containing bottle, and placing the solution containing 20 × 1010Placing single cell suspension of each cell into a cell accommodating bottle, placing 350L of DMEM/F12 culture medium into a culture medium accommodating bottle, placing 3D microcapsule dissolving agent into the dissolving agent accommodating bottle, controlling a starting filtrate/fluid infusion auxiliary pump in the device, introducing the single cell suspension and the swollen 3D microcapsules in the cell accommodating bottle and the 3D microcapsule accommodating bottle into a 500L bioreactor through a first connecting pipe for inoculation, detecting whether inoculation is successful or not through aseptic sampling fracture sampling in the inoculation process, introducing DMEM/F12 culture medium in the culture medium accommodating bottle through the first connecting pipe for cell amplification culture after inoculation is successful, introducing the 3D microcapsule dissolving agent through the first connecting pipe for de-encapsulation after amplification is completed, starting a magnetic suspension centrifugal sample introduction pump after de-encapsulation is successful, introducing the contents of the bioreactor into a filter body for circular filtration, the waste liquid penetrates through the filtering membrane in the filter body and enters the waste liquid tank along the second connecting pipe and the waste liquid pipe, and the amplified fine waste liquid enters the waste liquid tankThe cells are kept in a bioreactor, frozen stock solution is added into the amplified cells, then the cells can be introduced into an automatic filling machine for filling, and the filled cells are stored in a liquid nitrogen environment.
Comparative example 1
This comparative example provides a large-scale stem cell culture method, which is carried out by the apparatus shown in FIG. 1; the culture method comprises the following steps: carrying out cell culture on the Singapore ESCO commercialized paper carriers according to instructions;
the mesenchymal stem cells were inoculated into a commercial paper sheet carrier of ESCO singapore according to the product instructions and amplified using the equipment shown in fig. 1; then the corresponding dissolving agent is used for carrying out the capsulizing; harvesting the expanded cells;
wherein, the device shown in figure 1 comprises a bioreactor, a hollow fiber filter, a control device and a waste liquid bottle, the control device comprises a control host, a magnetic suspension centrifugal sample introduction pump and a filtrate/liquid supplement auxiliary pump which are integrated into a whole, the hollow fiber filter is used for filtering waste liquid and intercepting cells in the bioreactor, the bioreactor is respectively connected with a cell containing bottle, a culture medium containing bottle, a Singapore ESCO commercialized paper sheet carrier containing bottle and a lytic agent containing bottle through a first connecting pipe, and is connected with a central control fiber filter through a sample introduction pipeline and a return pipeline, the hollow fiber filter is connected with the magnetic suspension centrifugal sample introduction pump and the filtrate/liquid supplement auxiliary pump through a second connecting pipe, the magnetic suspension centrifugal sample introduction pump is connected with the waste liquid bottle through a waste liquid pipe, the hollow fiber filter comprises a filter body and an ultrasonic reflux flowmeter arranged on the filter body, the bottom of filter body links to each other with advance kind pressure sensor appearance pipeline through magnetic suspension pump head respectively, is equipped with backward flow pressure sensor and aseptic sample fracture on being close to the back flow way of filter body in proper order, advances kind pressure sensor and backward flow pressure sensor and is connected with the main control system, and the one end that the second connecting pipe of being connected with magnetic suspension centrifugation sampling pump is close to filter body is equipped with filtrating pressure sensor, and filtrating pressure sensor is connected with the main control system, and bioreactor still is connected with automatic filling machine.
Comparative example 2
This comparative example provides a large-scale stem cell culture method, which is carried out by the apparatus shown in FIG. 1; the culture method comprises the following steps: swelling the 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter is 0.5, and the pore diameter of the microcapsule is 10 nm;
inoculating mesenchymal stem cells into the swelled 3D microcapsules, wherein the inoculation density of the stem cells is 1 multiplied by 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: performing static culture for 1 day, adjusting the stirring speed to 30RPM during the static culture, adjusting the pH to 6.5 and DO 50% and the temperature to 37 ℃, supplementing the culture medium every 2 days, performing perfusion culture for 3 days, increasing the perfusion flow rate from 0.8vvd to 4vvd during the perfusion culture, adjusting the stirring speed to 150RPM, and supplementing the culture medium every 2 days;
3D microcapsule dissolving agent is used for carrying out the capsulizing;
harvesting the expanded cells;
inoculating the single cell suspension to the swelled 3D microcapsule by adopting a rotary culture mode, wherein the stirring speed of the rotary culture is 30 r/min;
the specific operation of swelling the 3D microcapsule, the preparation method of the 3D microcapsule, the composition of the 3D microcapsule dissolving agent, the equipment and the operation flow are the same as those of example 1.
Comparative example 3
This comparative example provides a large-scale stem cell culture method, which is carried out by the apparatus shown in FIG. 1; the culture method comprises the following steps: swelling the 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter is 0.5, and the pore diameter of the microcapsule is 10 nm;
inoculating mesenchymal stem cells into the swelled 3D microcapsules, wherein the inoculation density of the stem cells is 1 multiplied by 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: performing static culture for 1 day, adjusting the stirring speed to 80RPM during the static culture, adjusting the pH to 7.5 and DO 50%, adjusting the temperature to 37 ℃, supplementing the culture medium every 2 days, performing perfusion culture for 3 days, increasing the perfusion flow rate from 0.3vvd to 1vvd during the perfusion culture, adjusting the stirring speed to be unchanged, and supplementing the culture medium every 2 days;
3D microcapsule dissolving agent is used for carrying out the capsulizing;
harvesting the expanded cells;
inoculating the single cell suspension to the swelled 3D microcapsule by adopting a rotary culture mode, wherein the stirring speed of the rotary culture is 80 r/min;
the specific operation of swelling the 3D microcapsule, the preparation method of the 3D microcapsule, the composition of the 3D microcapsule dissolving agent, the equipment and the operation flow are the same as those of example 1.
Test example 1
The method and the equipment provided by the embodiments 1-3 and the comparative examples 1-3 of the invention are used for culturing the mesenchymal stem cells, wherein the cell inoculation amount of the comparative examples 1-3 is the same as that of the embodiment 1; detecting the survival rate and proliferation multiple of the harvested cells after each group of cells are cultured 7 days later; the cell proliferation multiple can be measured by trypan blue counting method, and the specific method comprises the following steps: cracking the microcapsule loaded with cells, fixing the volume, putting 0.5ml of cell suspension into a small test tube, adding 0.5ml of 0.4% of Taiwan phenol blue staining solution, lightly blowing and uniformly mixing by using a suction tube, staining for 3 minutes, then shaking the suspension uniformly, sucking the suspension liquid to drop on a blood counting chamber, wherein the blue cells are dead cells; the cell viability, which is (total number of cells-number of dead cells)/total number of cells, was calculated according to the following formula, and the results of the experiment are shown in table 1.
TABLE 1 results of cell viability test by each set of methods.
As can be seen from table 1, when the harvest time is 7 days, the proliferation fold and the cell viability rate of the method of examples 1 to 3 are higher than those of the comparative examples 1 to 3, and thus, the method for culturing stem cells in large scale provided by the present invention can significantly improve the cell viability rate, the cell proliferation fold and the cell harvest rate, significantly shorten the cell harvest time, and significantly reduce the culture effect of stem cells in large scale culture when other microcarriers are used to replace the 3D microcapsules of the present invention or specific parameters in the method provided by the present invention are modified.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (5)
1. A large-scale stem cell culture method is characterized by mainly comprising the following steps:
swelling a 3D microcapsule, wherein the 3D microcapsule is honeycomb-shaped, the diameter of the 3D microcapsule is 0.5-10mm, and the aperture of the 3D microcapsule is 10-10000 nm;
inoculating the single cell suspension into the swelled 3D microcapsule, wherein the inoculation density of the stem cells is 1-8 multiplied by 105And (4) performing amplification culture on the stem cells by using an amplification culture medium under the following culture conditions: static culture is carried out for 1-3 days, the stirring speed during the static culture is adjusted to 50-70RPM, the pH is adjusted to 7.0 and DO50 percent, the temperature is 37 +/-1 ℃, the culture medium is supplemented every 2 days, perfusion culture is carried out for 3-5 days, the perfusion flow rate during the perfusion culture is increased from 0.8vvd to 2vvd, the increasing speed of the perfusion flow rate is 1-3vvd/h, the stirring speed is adjusted to 100RPM, and the culture medium is supplemented every 2 days;
3D microcapsule dissolving agent is used for carrying out the capsulizing;
harvesting the expanded cells;
the preparation method of the 3D microcapsule comprises the following steps:
s1: uniformly mixing 1-6 wt% of sodium alginate solution and 0.1-5 wt% of gelatin solution in a ratio of 0.1-2:0.1-2 to prepare a first mixed solution;
s2: adding a foaming agent into the first mixed solution, and uniformly mixing, wherein the concentration of the foaming agent in the first mixed solution is 5-100g/L, so as to prepare a second mixed solution;
s3: dripping CaCl into the second mixed solution2Solution of said CaCl2The volume ratio of the solution to the first mixed solution is 1-2:1-2, and a 3D microcapsule solution is prepared;
s4: washing the 3D microcapsule solution with normal saline for 3-6 times, and then carrying out vacuum drying to obtain a dried 3D microcapsule;
s5: flattening and sterilizing the dried 3D microcapsule to obtain the 3D microcapsule.
2. The large-scale stem cell culture method of claim 1, wherein the 3D microcapsules after swelling are inoculated with the single cell suspension by means of rotary culture, wherein the stirring speed of the rotary culture is 30-50 r/min.
3. The large scale stem cell culture method of claim 1, wherein swelling the 3D microcapsule is performed by: stirring the 3D microcapsules and a DMEM/F12 culture medium uniformly, keeping the concentration of the 3D microcapsules in the DMEM/F12 culture medium at 100-1000g/L, standing for 3-5h, and transferring the 3D microcapsules into a DMEM/F12 culture medium containing 10% FBS, wherein the volume ratio of the DMEM/F12 culture medium containing 10% FBS to the DMEM/F12 culture medium is 1-2: 1-2.
4. The large-scale stem cell culture method according to claim 1, wherein the 3D microcapsule lysing agent comprises amino acids + citric acid + sodium citrate + EDTA sodium salt in a mass ratio of 0.1-1.0:0.1-2.0:0.1-1.0:0.1-2.0, the amino acids including any one of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, and histidine.
5. The large scale stem cell culture method of claim 1, wherein the volume ratio of the amount of the lytic agent to the volume of the swollen 3D microcapsule is 1-2: 1-4.
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Application publication date: 20200710 Assignee: Tang Yi Huikang (Shenzhen) Biomedical Technology Co.,Ltd. Assignor: Beijing tangyihuikang Biomedical Technology Co.,Ltd.|BEIJING ZHENXIGU MEDICAL RESEARCH CENTER (LIMITED PARTNERSHIP) Contract record no.: X2024980006120 Denomination of invention: Large scale stem cell culture methods Granted publication date: 20211126 License type: Common License Record date: 20240522 |